MXPA05002318A - Analytical method for the determination of infant formula protein digestibility in vitro. - Google Patents

Abstract

A method for in vitro determination of the digestibility of proteins in a nutritional product. The method utilized gastric and intestinal enzymes that are standardized for in vitro digestion process that mimics the in vivo digestive process. Further, specificity in digestion is determined by an amino acid profile.

Description

ANALYTICAL METHOD FOR THE DETERMINATION OF DIGESTIBILITY OF PROTEIN IN FORMULA FOR INFANTS IN VITRO FIELD OF THE INVENTION The invention relates to a method for quantifying the digestibility of protein in formula for infants. More particularly, the present invention relates to the determination of protein digestibility by analysis of amino acid configuration of total and soluble parts of an infant formula sample digested in vitro with USP digestive enzymes.

BACKGROUND OF THE INVENTION Infant formulas are commercially available in a variety of forms including powdered and concentrated liquid forms, ready to be eaten. Infant formulas typically contain casein and / or seroproteins intended to ensure that the child fed the formula receives adequate amounts of amino acids and, in particular, the amino acids essential for proper nutrition. Two factors in determining the nutritional quality of food proteins are digestibility and bioavailability. Typically, these formulas contain a higher level of protein than the level found in human breast milk. Infant formulas are manufactured with higher levels of protein to account for the lower assumed digestibility of proteins. Studies of formulas for infants have shown that the processes used during the elaboration of these formulas have nutritional consequences such as decreased solubility and / or digestibility of the proteins in the formula. For example, heat treatment for extended periods of time that is used to produce ready-to-eat infant formula and concentrated liquid formulas has been shown to reduce protein digestibility. As a result of exposure to heat, the proteins are denatured or aggregat, possibly altering their digestibility. The treatment of milk at high temperatures has also been studied and has been shown to increase the reactions of amino acids with sugars known as Maillard reactions. It has been shown that these reactions reduce the bioavailability of amino acids by limiting the accessibility of proteolytic enzymes. The digestion of protein in vivo is a two-step process. The first stage is the exposure of the protein to the pre-digestive enzyme pepsin. The second stage includes hydrolysis with pancreatic enzymes. The evaluation of amino acid availability in vivo is difficult because the protein digestion products are rapidly carried, and absorbed by, the small intestine. Additionally, endogenous proteins can be presented and can be digested and absorbed at different speeds from the proteins ingested as food or in the form of a food supplement. Therefore, in vitro analyzes of protein digestibility have been developed. The evaluation of the digestibility of the formula for infants has been carried out by colorimetric analysis of enzymatic hydrolysis (degree of hydrolysis using TNBS) and size exclusion chromatography techniques such as high performance liquid chromatography (HPLC). The accuracy and precision of the information provided by these procedures is compromised by the presence of insoluble protein and / or by chromatographic and spectrophotometric interferences. In vivo digestions of the infant formula have been conducted using the pre-digestive enzyme, pepsin, and pancreatin. Protein digestibility was determined by measuring the increased level of non-protein nitrogen (NPN) following the in vitro digestion process as determined by the Kjeldahl analysis. However, the enzymes used in these studies included the non-standardization of the enzyme activity and therefore the activity of the enzymes used in the digestion can vary significantly from batch to batch. In addition, nitrogen analysis by Kjeldahl procedures lacks specificity in the quantification of digested and undigested protein. The specificity in the types of digested amino acids provides better conduction so that the required formulations of nutritious products, including the formula for infants, ensure more digestion and absorption of essential amino acids by infants who are fed with the formula.

Digestive studies have included assays to determine the activity levels of proteins such as pepsin, trypsin and chymotrypsin used in the digestion process. The activity of pepsin has been measured in terms of units of products soluble in trichloroacetic acid (TCA). The activities of chymotrypsin and trypsin have been measured in terms of hydrolysis rate for a particular amino acid. While the level of activity of the enzymes to be used is determined, the standardization of such assays could be improved, the aggregated steps to determine enzyme activity are cumbersome and time-consuming. There is a need for a method for the determination of live protein digestibility that uses enzymes that have standardized activity. There is also a need for results of such a method to provide specificity in amino acid digestibility. The invention provides a method for determining protein digestibility while providing specificity in the quantification of total and digested protein (digested and undigested).

BRIEF DESCRIPTION OF THE INVENTION A method of the invention is a method for determining the digestibility of proteins, the method includes the steps of digesting a sample of a nutritive product and a reactive target with at least one enzyme; finish the digestion process; determining the total concentration of each of a plurality of amino acids for the sample and the blank; determine the total tryptophan concentration for the sample and the target; determining the soluble concentration of each of the plurality of amino acids for the sample and the blank; determine the soluble concentration of tryptophan for the sample and the target; and calculate the percentage of soluble amino acids in the digested sample of nutritional product. In another embodiment, the method comprises the steps of separating each digested sample and the blank in a first part and a second part; determining the total concentration of each of the plurality of amino acids for the first part of the sample and the first part of the blank; determine the total tryptophan concentration for the first part of the sample and the first part of the target; separating each of the second part of the sample and the second part of the target in a liquid phase and a solid phase; determining a soluble concentration of each of the plurality of amino acids in the liquid phase; and determining a soluble concentration of tryptophan in the liquid phase. In another embodiment, the separation step is selected from the group consisting of acidification, precipitation, centrifugation, filtration, and a combination of centrifugation and filtration. In another embodiment, the step to calculate the percentage of soluble amino acids includes the steps of adding the total concentrations of the plurality of amino acids and the tryptophan concentrations for the sample and blank; determining the difference in the total concentration of the plurality of amino acids and tryptophan between the sample and the blank; add the soluble concentration of the plurality of amino acids and the concentration of tryptophan in the sample and the blank; determining the difference in the soluble concentration of the plurality of amino acids and tryptophan concentration between the sample and the blank; divide the difference in soluble concentrations by the difference in the total concentrations to determine a coefficient; and multiply the coefficient by 100. In one modality, the nutritional product is formula for infants. In one embodiment, the digestion step uses one or more enzymes intended to mimic the environment of a human gastrointestinal tract. In a further embodiment, the enzymes are selected from the group consisting of pepsin, peptidases, pancreatin proteinase, papain, trypsin and quimo.trypsin. Still in a further embodiment, the stage of digestion of the sample includes the steps of obtaining the sample of nutritional product; adjust the pH to about 4.5; add pepsin; incubate the sample; increase the pH to about 7.0; add pancreatin proteinase; and incubate the sample. In a further embodiment, the step of finishing the digestion is to immerse the sample in a boiling water bath.

DETAILED DESCRIPTION OF THE INVENTION Proteins for which digestibility can be determined according to the present invention, can be found in various forms, including but not limited to, nutritional products, food supplements, pharmaceuticals or other products. They can be used at any age, for example, by infants, children or adults. They can be of particular value when used during periods of rapid growth, such as childhood, childhood and adolescence. The proteins for which it can determine the digestibility according to the invention, can be incorporated in a nutritious "vehicle or carrier" that includes but is not limited to the food categories of the FDA statute: conventional foods, foods for special food uses, food supplements and medical foods. Suitable sources of protein for nutritious products include milk, soy, rice, meat (eg, beef), animal and vegetable (eg, pea, potato), egg (egg albumen), gelatin, and fish. Suitable intact proteins include, but are not limited to, casein protein based on milk, based on soy, seroprotein, rice protein, beef collagen, pea protein, potato protein and mixtures thereof. Suitable protein hydrolysates also include, but are not limited to, soy protein hydrolyzate, casein protein hydrolyzate, seroprotein hydrolyzate, rice protein hydrolyzate, potato protein hydrolyzate, fish protein hydrolyzate, hyd rolisate of egg albumen, gelatin protein hldrolisate, a combination of vegetable and animal protein hydrolysates, and mixtures thereof. Hydrolyzed proteins (protein hydrolysates) are proteins that have been hydrolyzed or broken into shorter peptide fragments and amino acids. Such fragments of hydrolyzed peptide and free amino acids are easier to digest. In the broadest sense, a protein has been hydrolyzed when one or more amide bonds have been broken. The breaking of the amide bonds may originate incidentally or unintentionally during processing, for example, due to heating or tearing. For purposes of this invention, the term "hydrolyzed protein" means a protein that has been processed or treated in a manner intended to break amide bonds. Intentional hydrolysis can be effected, for example, by treating an intact protein with enzymes or acids. The hydrogenated proteins that are preferably used in the formulas according to this invention are hydrolyzed to such a degree that the ratio of amino nitrogen (AN) to total nitrogen (TN) varies from about 0.1 AN to 1.0 NT to about 0.4 AN. about 1.08 TN, preferably about 0.25 AN to 1.04 TN at about 0.4 AN to about 1.0 TN (given AN: TN ratios are for the single hydrolyzate protein source, and do not represent the given AN: TN ratios in the product of the final pediatric nutritional formula, since free amino acids can be added as a complement and could alter the reported value). The protein can also be provided in the form of free amino acids. A formula according to the invention is preferably supplemented with several free amino acids in order to provide a balanced and complete formula more nutritionally. The amino acids are the individual building blocks of protein biosynthesis. Non-essential amino acids are those that are synthesized in the ammonium body and several carbon sources. Non-essential amino acids include: Alanine (ALA), Serine (SER), Aspartic Acid (ASP), Glutamic Acid (G LU), Cysteine (CYS), Tyrosine (TYR), Asparagine (ASN), Proline (PRO), Glycine (GLY), and Glutamine (GLN). The abbreviation "GLX" refers to GLU plus GLN and the abbreviation "ASX" refers to ASP plus ASN. Essential amino acids are also required for the synthesis of live protein and must be obtained from food sources. These are Isoleucine (I LE), Leucine (LEU), Lysine (LYS), Methionine (MET), Phenylalanine (PH E), Threonine (TH R), Tryptophan (TRP), Valine (VAL), Histidine (H IS) and Arginine (ARG) (essential in young growth animals, but not in adults). Of the essential amino acids, tryptophan has the lowest daily intake requirement. Direct interference in protein digestibility can be extracted from analyzes that determine the type and concentration of amino acids in solution (the digestible or soluble part) after in vitro digestion and the level of amino acid concentration in the phase solid (the non-digestible part) after digestion. Examples of free amino acids suitable for addition to the formula include, but are not limited to, L-tryptophan, L-tyrosine, L-cysteine, L-taurine, L-methionine, L-arginine, and L-carnitine.

SOYA One component of the nutritional formula of this invention is soy protein. As described above, a number of sources of soy protein can be considered. The soy protein is isolated from the soybean seed. Soybeans are an excellent source of high quality protein where approximately 38% to 40% of the soybeans are protein. In summary (as shown in Esq. I), the processing of soybeans includes the extraction of oil from the cracked and dehusked soybeans that leave the defatted soybean flakes.

Scheme I Soybean Seed Processing Soybean Oil Floury SOYA Protein Concentrate SEEDS Defatted Soybean Flake Sees Protein Isolate Whey Protein Fiber The defatted soybean flakes are typically crushed in flours; alcohol-extracted or alcolin / H20 extracted to remove taste compounds and sugars to make protein concentrates; and processed with water to remove sugars and flavor compounds, precipitate and dry to make protein isolates. The protein and whey fiber are derivatives of the above products.

NUTRITIVE PRODUCTS Nutrient products contain macronutrients, ie, fats, proteins and carbohydrates, in varying relative amounts that depend on the age and condition of the intended user, and often contain micronutrients such as vitamins, minerals and trace minerals. The term "food" includes solids and liquids. The term "nutritional product" includes but is not limited to these FDA statutory food categories: conventional foods, foods for special nutritional uses, medical foods and formulas for infants. "Food for special nutritional uses" is intended to supplement a special nutritional need that exists because of a physical, physiological, or pathological condition by supplying nutrients to supplement the diet or as the sole item in the diet. A "medical food" is a food that is formulated to be consumed or administered enterically under the supervision of a physician and intended for the specific nutritional management of a disease or condition for which different nutritional requirements, based on scientific principles recognized, are established by medical evaluation.

In addition, a "food supplement" is a product intended to supplement the diet by ingestion in tablet, capsule or liquid form and is not represented to be used as a conventional food or as a single item of a meal or diet.

FÓMULAS FOR IN FANTES Infant formula refers to nutritive formulations that meet the standards and criteria of the Infant Formula Act, (21 USC §350 (a) et.seq.) And intended to replace or supplement milk. of human breast. Although such formulas are available in at least three different forms (powder, liquid concentrate and liquid ready-to-eat ("RTF"), it is conventional to talk about nutrient concentrations on a "how to feed" basis and Both the RTF is often described, it being understood that the other forms reconstitute or dilute according to the manufacturer's instructions essentially for the same composition and that one skilled in the art can calculate the composition relevant to powdered or concentrated forms. for "standard" or "term" infants refers to the formula for infants intended for infants who are born in full term as a first feeding.The protein, fat and carbohydrate components provide, respectively, from about 8 to 10, 46 to 50 and 41 to 44% of the calories, and the heat density varies closely from about 660 to about 700 kcal / L (or 19-21 Cal / fl.oz ), usually about 675 to 680 (20 Cal / fl.oz.). The distribution of calories between fat, protein and carbohydrate components may vary in some of different manufacturers of formula for infants in term. SIMILAC ™ (Ross Products Division, Abbott Laboratories), ENFAMIL ™ (Mead Johnson Nutritionals), and GOOD START ™ (Carnation) are examples of formula for infants on time. The "nutrient enriched" formula refers to formula for infants that is fortified in relation to the "standard" or "term" formula. The primary defining characteristic that differentiates nutrient-enriched formulas is caloric density; A secondary factor is the concentration of protein. For example, a formula with a heat density above about 700 Kcal / L or a protein concentration above about 18 g / L could be considered "nutrient enriched". Nutrient enriched formulas typically also contain higher levels of calcium (e.g., above about 650 mg / L) and / or phosphorus (e.g., above about 450 mg / L). Examples include Similac NEOSURE ™ and Sirmilac Special Care ™ formulas. The nutritive powders and liquids for which the digestibility of the present invention can be determined are made by generally conventional techniques known to those skilled in the art. In summary, three mixtures are prepared, mixed together, heat treated, standardized, spray dried (if applicable), packaged and sterilized (if applicable).

LIQUID PRODUCTS A carbohydrate / mineral mixture is prepared by first heating the water to a high temperature with stirring. Minerals are added like this. The minerals may include, but are not limited to, sodium citrate, sodium chloride, potassium citrate, potassium chloride, magnesium chloride, tricalcium phosphate, calcium carbonate, potassium iodide and trace mineral premix. A carbohydrate source, such as one or more of lactose, corn syrup solids, sucrose and / or maltodextrin is dissolved in the water, thereby forming a carbohydrate solution. A source of dietary fiber, such as soy polysaccharide, can also be added. The whole carbohydrate / mineral mixture is kept under stirring at elevated temperature until it is mixed with the other mixtures, preferably for no more than about twelve hours. A mixture of oil is prepared by combining and heating the basic oil mixture. The basic oil blend typically contains some combination of soy, coconut, palm olein, sunflower oil or tall oleic safflower and medium chain triglycerides. Emulsifiers, such as de-diacetyl tartaric acid esters, diglycerides, soybean, diglycerides, and soy lecithin, may be used. Any or all of the oil soluble vitamins A, D, E (form R, R, natural or synthetic) and K can be added individually or as part of a premix. Carotene Beta, which can function as an antioxidant in vivo, can also be irrigated, as can a stabilizer such as Irish moss. Oils containing LCPs important for this invention (eg, DHA and AA) can be added to the oil mixture. Care must be taken with these LCPs because they degrade easily and become rancid. The whole oil mixture is kept under agitation until it is mixed with the other mixtures, preferably for a period of no more than about twelve hours. A mixture of protein in water is prepared by first heating the water to a suitable elevated temperature with stirring. The protein source is thus added to the water with agitation. Typically, this protein source is from hydrolyzed or intact milk proteins (e.g., whey, casein), hydrolyzed or intact vegetable proteins (e.g., soy), free amino acids, and mixtures thereof. In general, any known source of amino nitrogen can be used in this invention. The whole protein mixture is kept under stirring at elevated temperature until it is mixed with the other mixtures, preferably for a period of no more than about two hours. As an alternative, some protein can be mixed in a protein-in-fat emulsion instead of protein in water.

The protein in water and the carbohydrate / mineral mixtures are mixed together with stirring and the resulting mixed mixture is maintained at an elevated temperature. After a brief delay (for example, a few minutes), the oil mixture is added to the mixed mixture of the preceding stage with stirring. As an alternative to the addition to the oil mixture, the LCP oils can be added directly to the mixture that results from the combination of the protein, carbohydrate / mineral mixtures and oil. After sufficient stirring to completely combine all the constituents, the pH of the complete mixture is adjusted to the desired range. The mixed mixture is then subjected to deaeration, ultra-high temperature heat treatment, emulsification and homogenization, then cooled to refrigerated temperature. Preferably, after the above steps have been completed, the appropriate analytical test for quality control is conducted. Based on the analytical results of the quality control tests, an appropriate amount of water is added to the agitated group for dilution. A vitamin solution, which contains water soluble vitamins and trace minerals (including sodium selenate), is prepared and added to the processed mixture with agitation. A separate solution containing nucleotides is prepared and also added to the processed mixture with stirring.

The pH of the final product can be adjusted again to achieve the optimum product stability. The completed product is thus filled in the appropriate metal, glass or plastic containers and subjected to terminal sterilization using conventional technology. Alternatively, the liquid product can be sterilized aseptically and filled into plastic containers.

DUST PRODUCTS A carbohydrate / mineral mixture is prepared as described above for the production of liquid product. An oil mixture is prepared as described above for the production of liquid product with the following exceptions: 1) Emulsifiers (mono, diglycerides, lecithin) and stabilizers (Irish moss) are typically not added to the powder, 2) In addition to beta carotene, other antioxidants, such as mixed tocopherols and ascorbyl palmitate, may be added to help maintain the oxidative quality of the product during any subsequent spray drying process, and 3) LCPs are added after the mixtures are mixed. , instead of the oil mixture. A mixture of protein in water is prepared as described above for the production of liquid product. The carbohydrate / mineral mixture, the protein mixture in water and the oil mixture are mixed together in a similar manner as described for the manufacture of liquid product. After pH adjustment of the completed mixture, the LCPs are thus added to the mixed mixture with stirring. Desirably, the LCPs are dosed slowly into the product as the mixture passes through a conduit at a constant rate before homogenization (inline mixing). After deareation, ultra-high temperature heat treatment, emulsification and homogenization, the processed mixture can be evaporated to increase the solids level of the mixture to facilitate more efficient spray drying. The mixture thus passes through a preheater and a high pressure pump and is spray dried using conventional spray drying technology. The spray-dried powder can be agglomerated, and then packaged in metal or plastic container or tinsel / laminate bags under vacuum, nitrogen, or other inert environment. Variations in any of these manufacturing processes are known or will be readily apparent to those skilled in the art. The invention is not intended to be limited to any particular processing process. The full text of all US Patents mentioned herein is incorporated for reference.

I NTESTIAL ENZYMES AND GASTRY CAS The enzymes used for the in vitro digestion described herein were produced in accordance with US Pharmacopoeia standards. The activity of the enzymes is therefore consistent from batch to batch. The United States Pharmacopeia (USP) is a nongovernmental organization that promotes public health by establishing state standards of the subject to ensure the quality of medicines and other health care technologies. These standards are developed through a process of public inclusion and are accepted worldwide. The standards developed by USP are published in the United States Pharmacopoeia and the National Form (USP-NF), which are recognized in the Federal Foods, Drugs and Cosmetics Act (21 U.S.C. §321 et seq.). Enzymes suitable for use in the method described herein include, but are not limited to, pepsin, peptidases, pancreatin proteinase, papain, trypsin and chymotrypsin.

In vivo Protein Digestion The digestive process in vivo is difficult to reproduce exactly. However, several conditions that exist in vivo can be reproduced in vitro. In vitro digestibility tests should be conducted under conditions that are as close as possible to in vivo conditions. For example, the pH and enzymes of the digestive system should be incorporated in the process of in vitro digestion. Equally, the in vitro digestion should be of a duration corresponding to the time that the proteins reside in the digestive tract. The in vitro digestion process described below mimics the pH and elaborates the intestinal and gastric enzymes of young infants. The time of in vivo digestion also mimics the time required for the food to pass through the digestive tract of young infants. Protein digestions of nutritious products were prepared by the following procedure: Prepare 80 ml of a nutritious product sample by obtaining a volume of nutritious product ready to be eaten, reconstitution of powder or dilution of liquid concentration. Transfer the suspension quantitatively into a 1 00 mL volumetric flask and dilute to 100 mL with water. (The suspension should be prepared in such a way that the 100 mL volumetric flask contains approximately 1.625 grams of protein, and such that the aliquot used with the pipette in the 20 mL screw cap bottle contains approximately 0.1625 grams of protein. of the test results depend, to some degree, on the use of a constant proportion of enzyme to the sample protein). Use a 10 mL pipette of the diluted sample in a 20 mL screw cap bottle. Adjust the pH of the aliquot from 10 mL to 4.5 with 1 M hydrochloric acid. Add 32 mg of Pepsin USP (U.S. Pharmacopeia, 12601 Twinbrook Parkway, Rockville, D 20852) and shake to completely suspend pepsin.

Incubate in the bottle at 37 ° C for thirty minutes. Raise the pH to 7.0 with 0.5M Na.HC03. Add 3.0 mL of freshly prepared suspension of Pancreatic Protease Amylase USP (U.S. Pharmacopeia, 12601 Twinbrook Parkway, Rockville, D 20852) at 25 mg / mL in 0.1 M NaHCO3. Shake to completely suspend the enzyme. Incubate the bottle at 37 ° C for sixty minutes. Immerse the bottle in a bath of boiling water for 4 minutes. Cool to room temperature. Transfer quantitatively the resulting digestion into a 25 mL volumetric flask with tar using the water to aid in the transfer. Dilute the digestion in the flask with water and record the weight. The reactive targets are digested throughout the nutrient samples for use in the calculation of the total soluble and total parts of the amino acids in the digested samples. Separate aliquots of digestion were taken to determine the amino acid configuration and tryptophan concentrations. However, if the amino acid configuration and tryptophan concentrations will be determined from the same aliquot of digestion, no separation steps are required.

Amino Acid Configuration Before testing the total amino acid configuration, 100 μ? of digestion was transferred by pipette into a 2 mL vial with tar, and the weight was recorded. 2.0 mL of 6M HCl were added and the vial was placed under nitrogen blanket, sealed and heated at 1 1 0 C for 22 hours. The digestion was evaporated in this way for dryness, then it was resuspended in 2 mL of Na-S regulator. The resuspension was filtered through a Gelman Acrodisc (0.45 um, Gelman P / N 4497). Before testing the soluble amino acid configuration, 10 mL of the digestion was transferred by pipette into a 50 mL centrifuge tube with tar. 10 mL of 24% trichloroacetic acid were added, the tube was capped and mixed well. The weight of the tube contents was recorded. The tube and the contents were centrifuged at 3000 times the gravity for thirty minutes, the liquid was thus filtered through Whatman No. 41 paper. 200 μ? of the filtrate was transferred by pipette into a 2 mL ampule with tar, and the weight of the sample was recorded. As with the total amino acid test, 2.0 mL of 6M HCl were added and the vial was placed under nitrogen blanket, sealed and heated at 1 10 0 C for 22 hours. The digestion was thus evaporated for drying, then resuspended in 2 mL of Na-S regulator. The resuspension was filtered through a Gelman Acrodisc (0.45 um, Gelman P / N 4497). If the total and soluble amino acid configurations are determined from the same digestion aliquot, a separation step (such as a centrifugation) is not required. The proteins are hydrolyzed to their constituent amino acids by acid hydrolysis, and the acid hydrolyzate is tested by an automatic amino acid analyzer (Model 6300 Amino Acid Analyzer, Beckmann Instruments, I nc., Palo Alto, CA). The analyzer uses ion exchange chromatography to separate the individual amino acids, and the post-column derivation with ninhydrin to generate amino acid derivatives that are thus detected and quantified by a colorimeter.

Determination of Triptofan The determinations of tryptophan were made by the method described by S. E. García and J .H. Baxter in Determination of Trytophan Contained in Infant Formulas and Medical Nutritionals, J AOAC Int 1 992 Nov-Dec; 75 (6): 1 1 1 2-9, incorporated herein by reference. For the determination of tryptophan of the total protein, 3.0 mL of digestion was transferred to a 50 mL volumetric flask. 1.0 mL of pronase solution (1.7 mg / mL in 0.05 M TRIS, pH 7.5) was added and the volume was diluted to 50 mL with a pH 7.5 regulator. The 50 mL solution was thus incubated at 50 ° C for six hours. The tryptophan was determined thus by H PLC procedure. For the determination of triptofan of soluble protein6.0 mL of the filtrate as prepared for the determination of soluble amino acid was transferred by pipette to a 50 mL beaker. 30 mL of 0.05M TRIS, pH at 7.5, were added. The pH was adjusted to 7.5 using 45% potassium hydroxide solution. As with the tryptophan test for total protein, 1.0 mL of pronase solution (1.7 mg / mL in 0.05M TRIS, pH 7.5) was added and the volume was diluted to 50 mL with pH regulator. 7.5. The 50 mL solution was thus incubated at 50 ° C for six hours. The tryptophan was determined thus by HPLC method.

HPLC System for the Determination of Triptofan Column: YMC ODS-AQ, 4.6 x 250 mm, 120A, 5um, Waters # AQ12S052546WT. Mobile Phase A: 900 mL, 0.02 M KH2P04, 100 mL of acetonitrile; pH 3.1 with H3P04. Mobile Phase B: 200 mL of laboratory water, 800 mL of acetonitrile. Flow rate: 0.5 mL / minute. Temperature: 20 ° C. Detection: UV at 280 nm, 214 nm. Injection: 1 0 μ? Execution time: 50 minutes Avoidance program: 0% B of 0-5 minutes, 0-25% B of 5-34 minutes, 25-100% of B of 34-35 minutes, 100% of B of 35 -37 minutes, 1 00-0% of B of 37-38 minutes. Standard solutions: Abbott Laboratories PPD L-Tryptophan at approximately 28 mg / L (High Standard) approximately 14 mg / L (Medium Standard), and approximately 7 mg / L (Low Standard) in laboratory water.

Calculation of Protein Digestibility Add concentrations of amino acid configuration and tryptophan concentration for Total Protein of Reactive White. Designate these values as "RT". Add the concentrations of amino acid configuration and tryptophan concentration to the Soluble Protein of Reactive White. Designate this value as "RS". Ag irrigate the concentrations of amino acid configuration and tryptophan concentration obtained for the Total Protein of Sample Digestion. Designate this value as "IT". Add the concentrations of amino acid configuration and tryptophan concentration obtained for the Soluble Protein of Sample Digestion. Designate this value as "I S". Calculate Protein Digestibility as follows: Protein Digestibility IS - RS x 1 00 IT-RT All references, including publications, patent applications, and patents, cited herein are incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated for reference and established in its entirety in the present.

The use of the terms "a" and "an" and "the" and similar references in the context of the description of the invention (especially in the context of the following claims) will be construed to cover both the singular and the plural , unless otherwise indicated or contraindicated clearly by context. The recitation of ranges of values herein is intended merely to serve as a short method to refer individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated in the specification as if it will be cited individually in the present. All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contraindicated in another way by context. The use of any and all examples, or exemplary language (eg, "such as") provided herein, is intended to merely illuminate the invention merely and not to have a limitation on the scope of the invention unless it is claim in another way. No language in the specification shall be construed as indicating any unclaimed element as essential as the practice of the invention. While some advantages and potential objects have been expressly identified herein, it should be understood that some embodiments of the invention can not provide all, or any, of the advantages and objects expressly identified.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to employ such variations as suitable, and the inventors intend for the invention to be practiced otherwise than specifically described herein. According to the above, this invention includes all modifications and equivalents of subject matter recited in the appended claims thereto as permitted by applicable law. In addition, any combination of the above-described elements in all possible variations thereof is understood by the invention unless otherwise indicated herein or otherwise clearly contraindicated by the context.

EXAMPLES The following examples further illustrate the invention but, of course, should not be construed in any way as limiting its scope.

EXAMPLE 1 Three hydrolysates of Arla powder serology (WPH), Alternating Arla WPH 1, 2 and 3, were obtained from Arla Food Ingredients, both Nr. Vium DK-6920 Videbaek Denmark. 80 ml_ of reconstituted powdered WPHs, containing approximately 1,625 grams of protein and 80 ml of reconstituted GOOD START ™ Nestle, also containing approximately 1,625 grams of protein, were digested in vitro together with a reactive blank according to the process described herein. The amino acid configuration and tryptophan concentrations were determined for the total digestion and the soluble part of the digestion as described herein. The amino acid configurations are shown in Table 1. A comparison of protein digestibility is shown in Table 2.

Table 1. Amino Acid Configuration of the Digested Samples of Example 1. The concentration is found in grams of amino acid per 100 grams of total protein. Amino Acid Arla 1 Arla 2 Arla 3 GOOD STARTIIVI Nestle ASX 9.70 11.7 10.1 10.3 THR 6.57 7.73 6.87 7.11 SER 4.46 5.21 4.64 4.69 GLX 16.2 19.6 17.0 16.7 PRO 5.73 6.88 5.97 5.83 5.93 GLY 1.34 1.64 1.33 1.32 1.32 WING 4.46 5.22 4.69 4.74 CYS 1.66 2.03 1.81 0.99 VAL 4.97 5.90 5.54 5.34 MET 1.64 1.87 1.66 1.69 ILE 5.38 6.45 6.11 6.26 LEE 8.84 10.2 9.88 9.40 TYR 2.40 2.59 2.63 2.31 PHE 2.64 2.91 3.02 2.76 H1S 1.70 1.94 1.82 1.82 1.76 LYS 8.78 10.3 9.06 8.69 ARG 2.07 2.44 2.26 2.06 TRP 1.36 1.27 1.50 1.22 TOTAL 89.9 105.9 95.9 93.2 Note: TRP was determined by HPLC. These values are based on the total protein concentrations of 77.0%, 78.0%, 78.5%, and 12.3% for Arla 1, Arla 2, Arla 3 and GOOD START ™ Powder, respectively.

Table 2. Comparison of Protein Digestibility Product of Soluble Protein Seroprotein after Digestion (as grams of amino acid per 100 grams of protein *) GOOD START IM Nestle 93.2 Arla WPH Alternate 1 89.9 Arla WPH Alternate 2 105.9 Arla WPH Alternate 3 95.9 * that is, as grams of total amino acids in soluble protein per 1 00 grams of total sample protein.

Because the WPH digestibility can be reduced by the process of formula making for infants, the definitive conclusions regarding the digestibility of this nutrient product are best extracted from comparisons of formulas for infants.

Example 2 Three samples of S I MI LAC ™ with low iron and three samples of GOOD START ™ Nestle powder were each reconstituted with water and 80 ml_ of each reconstitution contained approximately 1.625 g protein bouquets. The formula samples were digested in vitro together with a reactive blank according to the process described herein. The in vitro digestion process described early imitates the digestive system of young infants in the parameters of pH, enzymes present, and time of digestion. The amino acid configuration and the tryptophan concentrations were determined for the total digestion and the soluble part of the digestion as described herein. An example of an amino acid configuration is shown in Table 3. The calculations of protein digestibility as described herein were conducted. A comparison of protein digestibility is shown in Table 4.

Table 4. Amino Acid Configuration of the Digested Samples of Example 2. The concentrations are as mg / L of Sample Suspension. White Reagent Formula for Infants Amino Acid Protein Protein Protein Total Protein Total Soluble Soluble ASX 430 375 1914 1703 THR 161 1 34 1065 939 SER 204 173 1082 981 GLX 434 391 3693 3428 PRO 152 130 1562 1232 GLY 242 209 558 462 ALA 167 141 825 739 CYS 45 29 181 136 VAL 170 137 121 8 1003 MET 34 27 407 372 I LE 142 1 13 1 107 954 LEU 182 143 1861 1580 TYR 150 127 850 757 PHE 1 12 90 825 650 HIS 88 75 496 420 LYS 237 218 1613 1533 ARG 224 205 733 676 TRP 85 69 306 244 TOTAL 3259 2786 20,296 17,809 Protein Digestibility = 17,809 - 2,786 x 100 = 88.2% 20,296 - 3,259 Table 4. SIMILAC ™ Protein Digestibility Conversion GOOD START ™ Nestle Powders. Formula for Infants Protein Digestibility in vitro GOOD START ™ Nestle Powder 89.0% +/- 1.2% (n = 3) # 107EWGS0159S SIMILAR I with Iron Powder 87.8% +/- 0.6% (n = 3) Low # 61927RE

Claims (1)

1. CLAIMS 1. A method for determining protein digestibility, the method comprising the steps of: digesting a sample of a nutritive product and a reactive blank with at least one enzyme; finish the digestion process; determining the total concentration of each of a plurality of amino acids for the sample and the blank; determine the total tryptophan concentration for the sample and the target; determining the soluble concentration of each of the plurality of amino acids for the sample and the blank; determine the soluble concentration of tryptophan for the sample and the target; and calculate the percentage of soluble amino acids in the digested sample of nutritional product. The method according to claim 1, further comprising the steps of: separating each digested sample and blank in a first part and a second part; determining the total concentration of each of the plurality of amino acids for the first part of the sample and the first part of the blank; determine the total tryptophan concentration for the first part of the sample and the first part of the target; separating each of the second part of the sample and the second part of the target in a liquid phase and a solid phase; determining a soluble concentration of each of the plurality of amino acids in the liquid phase; and determining a soluble concentration of tryptophan in the liquid phase. The method according to claim 2, characterized in that the separation step is selected from the group consisting of acidification, precipitation, centrifugation, filtration, and a combination of centrifugation and filtration. The method according to claim 1, characterized in that the step for calculating the percentage of soluble amino acids comprises: adding the total concentrations of the plurality of amino acids and the concentrations of tryptophan for the sample and the blank; determining the difference in the total concentration of the plurality of amino acids and tryptophan between the sample and the blank; add the soluble concentration of the plurality of amino acids and the concentration of tryptophan in the sample and the blank; determining the difference in the soluble concentration of the plurality of amino acids and tryptophan concentrations between the sample and the blank; divide the difference in soluble concentrations by the difference in the total concentrations to determine a coefficient; and multiplying the coefficient by 100, 5. The method according to claim 1, characterized in that the nutritional product is formula for infants. The method according to claim 1, characterized in that the digestion step uses one or more enzymes intended to mimic the environment of a human gastrointestinal tract. The method according to claim 6, characterized in that the enzymes are selected from the group consisting of pepsin, peptidases, pancreatin proteinase, pancreatic protease amylase, papain, trypsin and chymotrypsin 8. The method according to claim 1, characterized in that the The digestion stage of the sample comprises the steps of: obtaining the sample of nutritional product; adjust the pH to about 4.5; add pepsin; incubate the sample; increase the pH to about 7.0; add pancreatin proteinase; and incubate the sample. The method according to claim 8, characterized in that the pepsin is prepared by US Pharmacopoeia standards for enzymatic activity. The method according to claim 8, characterized in that the pancreatin proteinase is prepared by US Pharmacopoeia standards for enzymatic activity. eleven . The method according to claim 1, characterized in that the step of finishing the digestion comprises immersing the sample in a boiling water bath. The method according to claim 1, characterized in that at least one enzyme is an intestinal or gastric enzyme prepared by US Pharmacopoeia standards for enzymatic activity.